JOURNAL OF BONE AND MINERAL RESEARCH

Volume 17, Number 8, 2002

2002 American Society for Bone and Mineral Research

In Vitro Engineering of Human Autogenous Cartilage

URSULA ANDERER and JEANETTE LIBERA

ABSTRACT
A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in
vitro engineered cartilage, such as prevention of immune response and structural as well as functional
integration to surrounding tissue, we established a three-dimensional cell culture system without adding
exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology
and protein expression was analyzed using histological and immunohistochemical investigations on spheroid
cryosections. A cartilage-like tissue similar to naturally occurring cartilage was generated when spheroids
were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the
synthesis of the hyaline-specific proteins collagen type II and S-100, as well as the synthesis of hyaline-specific
mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior
of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids
quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics
to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human
serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim
of cells remained on the surface. These results show that the spheroid culture allows for the first time the
autogenous in vitro engineering of human cartilage-like tissue where medium supplements were restricted to
human serum. (J Bone Miner Res 2002;17:1420 1429)
Key words:

tissue engineering, cartilage, autologous chondrocyte transplantation, in vitro, threedimensional

INTRODUCTION
ELF-REPAIR OF human hyaline cartilage does not occur.
Therefore, cartilage injuries initiate a progressive degradation that eventually results in osteoarthritis.(13) An
accepted approach for the regeneration of hyaline cartilage
after traumatic cartilage damage is the autologous chondrocyte transplantation.(4 6) However, the in vitro engineering
of three-dimensional hyaline cartilage tissue with the respective structure and function is still a challenge for cartilage repair in contrast to using cell suspensions as transplants. For three-dimensional in vitro engineering, cellseeded scaffolds have been tested. The in vitro culturing of

The authors have no conflict of interest.

chondrocytes in the presence of growth factors on various

three-dimensional scaffolds resulted in the maintenance of
the cartilage-specific phenotype.(79) In animal models,
these cell-seeded scaffolds allowed a formation of repair
tissue similar to hyaline cartilage.(8,10,11) Unfortunately, the
repair is often accompanied by considerable fibrocartilage
formation.(12,13) Further demands on in vitro engineered
tissues are the integration into native tissue and the tissue
formationadapted resorption of scaffolds. However, neither the integration of these cell-seeded scaffolds into surrounding host cartilage, nor predictable resorption of the
scaffold polymers, has been optimized for application in
patients.(14)
Few approaches have been advanced to overcome these
problems by renouncing any scaffolds. The generation of

co.don AG, Molecular Medicine, Biotechnology, and Tissue Engineering, Teltow, Germany.

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IN VITRO ENGINEERING OF HUMAN AUTOGENOUS CARTILAGE

cartilage-like structures was observed when human chondrocytes isolated from osteoarthritic hips were cultured in a
gyratory shaker.(15) However, to maintain the differentiated
phenotype, the addition of growth factors was necessary.
The aim of this study was to engineer three-dimensional
hyaline human cartilage that has the capacity to integrate
with native cartilage and prevent immune responses. For
that purpose, we established an autologous spheroid system
to culture human chondrocytes without adding xenogenous
serum, growth factors, or scaffolds, considering that several
growth factors and scaffolds are not permitted for use in
humans. Only human serum was added to the cell culture
medium. In this autologous spheroid system, cells form
three-dimensional aggregates and generate their own extracellular matrix that is similar to the natural matrix of hyaline
cartilage. These in vitro engineered cartilage tissues adhere
to and integrate into native tissue. We further show that the
addition of fetal calf serum (FCS) or antibiotics to culture
medium delays or even inhibits the engineering of cartilagelike tissue in vitro. Our methodology describes, for the first
time, the in vitro engineering of three-dimensional autogenous cartilage that seems suitable for the treatment of cartilage lesions, degenerative changes in cartilage, and pharmaceutical test systems.

MATERIALS AND METHODS

Chondrocyte culture
Articular cartilage was obtained from human articular
condyles in volunteer patients undergoing knee surgery.
Cartilage (60 100 mg) was minced and digested in a 50-ml
Falcon tube using 20 25 U/mg collagenase type II (Biochrome, Berlin, Germany) at 37C for 8 h in a gyratory
shaker (110 rpm). Isolated cells were washed and resuspended in culture medium with the addition of FCS (n 2),
autologous serum (n 3), or pooled human serum (n 6)
from separate human volunteers. No growth factors, cytokines, or other supplements were added. Chondrocytes were
seeded in Falcon culture flasks (75 cm2, 15 ml medium) and
maintained at 37C in a humidified atmosphere and 5%
CO2. Medium was changed twice weekly. After reaching
confluence, the cells were trypsinized using trypsin-EDTA
(PAA-Laboratories GmbH, Co lbe, Germany) and cultured
in larger Falcon flasks (225 cm2, 35 ml medium). Experiments were performed with chondrocytes between the second and seventh monolayer passage. For generation of
spheroids, chondrocytes were seeded in hydrogel-coated
96-well plates. For hydrogel coating, agarose was melted in
cell culture medium (2% wt/vol) and pipetted into the wells.
After a jelling time of 2 h at room temperature, cell suspensions (1 105 and/or 2 105 cells/well in 250 l
medium) were added. Starting with 1 105 and 2 105
cells/well should hint at a possible change in size and
quality of cell aggregates. Cell aggregates of the appropriate
experimental set-ups were analyzed after 5 days, 2 weeks,
and 1, 2, and 3 months (n 2). After aggregation of
chondrocytes, 210 single spheroids were transferred into
one well, allowing coalesce of spheroids. For co-culture,
single spheroids were placed on cartilage tissue of isolated

1421

human femoral condyles (medial and lateral, 3.5 2 0.8

cm) from volunteer patients (n 3 patients) undergoing
knee replacement surgery because of osteoarthritis. Condyles with adherend spheroids were surrounded and covered
with medium (50 ml) and cultured in petri dishes under
standard conditions. After different time points (45 minutes,
3 weeks), condyles were frozen and histologically analyzed.
To assess cell growth and cell differentiation in the presence
of antibiotics, chondrocytes and spheroids were cultured in
the addition of penicillin (100 U/ml) and streptomycin (100
mg/ml).

Proliferation analysis
Cell proliferation was assessed by BrdU incorporation
into monolayer (incorporation time: 6 h, 24 h) and aggregated cells (incorporation time: 6 h, 24 h, 2 days) using a
cell proliferation kit (Amersham, Freiburg, Germany).
Three samples per time point were analyzed.

Histological analysis
To analyze chondrocytes in the monolayer culture, cell
suspensions were added to glass slides in appropriate dishes.
The cells adhere and proliferate directly on the glass surface. Slides were washed twice in phosphate buffered saline
(PBS) and fixed in methanol/acetone (1:2) at 20C for 10
minutes. Spheroid specimens were embedded in Tissue-Tek
(Miles, Naperville, IL, USA), snap-frozen in liquid nitrogen, and cut into 5- to 7-m sections using a cryomicrotome
(Microm, Walldorf, Germany). Sections were mounted on
pretreated slides (Superfrost Plus; Menzel Gla ser, Braunschweig, Germany), air dried, and fixed in concentrated
acetic acid:ethanol (1:20) at room temperature (RT) for 20
minutes. Hematoxylin/eosin (HE), safranin O, and Goldnertrichrome staining were performed on serial sections of
spheroids and monolayer cells directly grown on slides
using standard histochemical techniques.

Immunohistochemical analysis
Collagen and S-100 antigen(16) expression was assessed
on serial sections of snap-frozen spheroids and monolayer
slides using the avidin biotin complex (ABC) method
(DAKO, Hamburg, Germany). As primary antibodies, polyclonal rabbit antisera recognizing human types I and II
collagen (1:30 and 1:15; Novo Castra, Newcastle upon
Tyne, UK) and S-100 protein (1:100, DAKO) were used. To
avoid nonspecific binding of the antibodies, slides were first
incubated with tris(hydroxymethyl)aminomethane (TRIS)
buffer containing 5% normal porcine serum and 0.1% bovine serum albumin (BSA) at RT for 30 minutes. Incubation
with primary antisera followed at 4C in a humidified chamber for 12 h. After three washes in TRIS, bound primary
antibody was detected using the DAKO ESAB System
AP, with fuchsin as the substrate for alkaline phosphatase
(DAKO). Cell nuclei were counterstained with hematoxylin.
Control procedures paralleled each step. TRIS was applied to the sections instead of the primary antibodies as a

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ANDERER AND LIBERA

control for the secondary antibody. Articular cartilage-bone

sections were used as respective controls for the specificity
of the primary antibodies against collagen type I and II. For
the S-100 antiserum, a neuroblastoma cell line (SK-N-SH;
American Type Culture Collection, Rockville, MD, USA)
and fibroblasts were used as positive and negative controls,
respectively.

RESULTS
Spheroid formation
For in vitro engineering of three-dimensional hyaline
cartilage tissue, we investigated the differentiation of human chondrocytes in aggregate culture. After seeding chondrocytes in hydrogel-coated wells, cells aggregated to form
a disc measuring 900-1200 m in diameter after 1 day.
During the next 2 weeks, discs rounded and became more
compact, with diameters ranging from 350 to 500 m (Figs.
1A and 1B). Coalescence of several spheroids was initiated
by an active migration of surface cells of adjacent aggregates (Fig. 1B). Through 8 days, remodeling of the aggregates resulted in a more homogenous spherical structure,
and gaps between aggregates were filled (Fig. 1C). Separate
from the ability to merge, aggregates also migrated and
contacted tissue culture flasks or glass slides (Fig. 1D).

Spheroid morphology and differentiation

During the initial 2 weeks, spheroid chondrocytes produced high amounts of acidic mucopolysaccharides, indicated by the green intercellular matrix on Goldner-stained
sections (Fig. 2B). Aggregates cultured in medium with
autologous and pooled serum revealed a homogenous distribution of intact spherical cells in the interior and flattened
cells on the surface (Figs. 2A, 2B, and 3A3C). Viable cells
and round nuclei in the interior of spheroids suggest that
sufficient nutrient supply is available for all cells. As individual aggregates merged, an adhesion zone consisting of
several flattened cell layers between two aggregates was
still observable after 24 h in close contact (Fig. 2A, arrowhead). However, by 5 days, these cells had achieved a
histotypical morphology similar to that of internal cells
(Fig. 2A, arrow).
Chondrocytes in monolayer culture loose the expression
of collagen type II and the cartilage specific intracellular
protein S-100(16) completely during their third or fourth
passage, corresponding with a high expression of collagen
type I (data not shown). However, when chondrocytes of the
second to the seventh monolayer passage were cultured as
spheroids, a reexpression of collagen type II and S-100
occurred (Figs. 3A and 3B). This chondrocyte differentiation was accompanied by the loss of cell proliferation,

FIG. 1. Chondrocyte aggregates cultured in autologous serum after

different times. (A) Ball-shaped aggregate after 4 days. (B) Merge of
three 16-day-old aggregates after 2 days: cells stretching from one

aggregate to another (arrow). (C) Merged aggregates from B, 8 days

later: former gap is filled with cells (arrow). (D) Cell emigration from
aggregate to an artificial surface after 2 days. (AC) Live cell aggregates and (D) immunohistochemistry of collagen type I.

IN VITRO ENGINEERING OF HUMAN AUTOGENOUS CARTILAGE

1423

FIG. 2. Histological analysis of chondrocyte (seven passages in monolayer) aggregates cultured in human pool serum for 16 days. (A) HE
staining: difference in cell morphology in the inner (spherical cells) and outer part (flattened cells) of the aggregate. Flattened cells in the adhesion
zone after 1 day (arrowhead) and spherical cells after 5 days (arrow). Gap between two aggregates (two arrowheads) was filled with cells after
5 days (two arrows). (B) Goldner-stained sections of aggregates from A.

analyzed by BrdU incorporation into cell nuclei (data not

shown). In the presence of autologous serum, a weak expression of collagen type II in the outer cell layer of spheroids was detectable after 1 week. After 6 weeks, a weak
expression of collagen type II was also observed in the
interior (Fig. 3A). In medium supplemented with allogenous
pooled human serum, reexpression of collagen type II in the
outer cell layer was delayed and not as high as in autologous
culture. Collagen type II was detectable only after the third
week in culture (data not shown). The S-100 reexpression
was generally similar to that of collagen type II under
autologous and human pooled serum conditions (Fig. 3B).
In contrast to the high expression of collagen type I in all
cells in monolayer culture, a reduced expression or complete loss of collagen type I was found in the interior cells
of the aggregate (Fig. 3C). Throughout the time course of
three-dimensional culture, and independent of serum type,
the outer cell layers expressed the highest amount of collagen type I. The expression of collagen type I was higher
than that of collagen type II.
Under prolonged culture conditions, the in vitro generated tissues more closely resembled the typical features of
in vivo cartilage: solid and elastic aggregates with a bright
white surface (Fig. 4A). Cryosections revealed flattened
cells on the surface (Figs. 4B and 4E) and an increased
amount of intercellular matrix in the core of spheroids (Figs.
4B 4D). This core area stained positive with safranin O,
affirming the synthesis and extracellular deposition of hyaline cartilage specific proteoglycans (Fig. 4B). Cell number
per given matrix area varied with the depth in the aggregate.
In the core area, cells were widely separated by matrix, and
the cell number per given matrix area was approximately
twice that of native cartilage (Figs. 4B and 4F). Nearer the
surface, the number of cells per matrix area increased and
was twice that of the core. In contrast to the randomly
distributed cells in the core area of aggregates, cells on both
sides of a merging zone were oriented vertical to the contact
area (Fig. 4B). This orientation is known for native articular
cartilage where chondrocytes are oriented perpendicular to
the tidemark.

Influence of antibiotics and FCS on cell condition

Usually, antibiotics are added to cell culture medium. In
the presence of penicillin and streptomycin in chondrocyte
monolayer cultures with autologous serum, we observed a
prolongation of culture time to reach confluence compared
with the absence of antibiotics. Furthermore, the aggregation of chondrocytes was delayed for up to 5 days (data not
shown). During the first 4 weeks in aggregation culture, the
expression of cartilage-specific proteins and the cell density
were similar to aggregates cultured in the absence of antibiotics. However, there is no augmentation in the expression
of collagen type II, S-100, and acidic mucopolysaccharides
in aggregated chondrocytes up to 3 months (data not
shown). This parallels the still high and unchanged cell
density in spheroids cultured in the presence of antibiotics
observed after 3 months (data not shown).
While growing human chondrocytes in monolayer and
aggregate culture in addition of FCS, several differences
were apparent. In the monolayer, the proliferation rate of
chondrocytes decreased (data not shown). Furthermore,
when cells were transferred in aggregate culture, the
aggregation of cells was delayed significantly. By 7 days,
a central hole in the aggregate was present, and surface
cells were more densely packed (Fig. 5A). During the
first 6 weeks in aggregate culture with FCS, no quantitative differences in the expression of collagen type I and
polysaccharides compared with autologous cultured aggregates were found (data not shown). However, the
expression of collagen type II and S-100 protein was
delayed and reduced with only a weak expression after 4
weeks (Fig. 5B, shown for collagen type II). After 3
months in FCS culture, the inner part of the aggregates
was disintegrated and crumbled. HE staining revealed the
absence of viable cells. Only outer cells retained the
typical morphology and expressed type I collagen and
S-100 (Figs. 5C and 5D). However, cells and matrix were
loosely organized, and a shedding of the outer cells was
detected (Figs. 5C and 5D).

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ANDERER AND LIBERA

morphology from flattened to spherical cells. However, the

cartilage on the opposing side of the aggregate retained its
flattened surface cells (Fig. 6A). Over the course of 3 weeks
in culture, the spheroids became more flattened. By active
migration, aggregate cells were widely distributed on the
surface of the degenerated cartilage. The cells not only
migrated on the native cartilage surface but also synthesized
new matrix (Fig. 6B). The migratory capacity enabled chondrocytes to integrate in surface fissures in addition to covering the surface (Fig. 6B). No active invading growth of
spheroid derived cells was observed. The newly formed
matrix on the cartilage explant surface is characterized by
stronger HE staining (Fig. 6B). This layer shows a higher
cell density than native tissue, indicating the cartilage forming capacity of spheroids.

DISCUSSION

FIG. 3. Immunohistochemical analysis of chondrocyte (three passages in ML) aggregates cultured in autologous serum for 6 weeks. (A)
Collagen type II, (B) S-100, and (C) collagen type I cells were immunolocalized on frozen sections counterstained with hematoxylin.

Aggregate attachment
To assess the integrative capacity of in vitro engineered
tissues, a cartilage-explant spheroid co-culture system was
used. After only 45 minutes, multiple focal adhesion points
were formed connecting the in vitro generated tissue with
native cartilage (Fig. 6A). Surface cells of the 3-week-old
aggregates in the contact area had already changed their

This study shows the potential to use a three-dimensional,

autologous in vitro culture system to engineer human articular cartilage. The formation of three-dimensional cartilagelike tissue was achieved without using any scaffolds. The
only supplement to culture medium was patient-specific
serum. No growth factors or other additives were used to
induce chondrogenic differentiation and maintain long-term
stability of the tissue constructs. The engineered tissue
constructs attach, migrate, and integrate with native tissues,
thereby meeting important requirements for tissue reconstruction and/or regeneration.
For generating cartilage in vitro that typifies not only the
morphology but also sustains the physiological behavior of
chondrocytes, it is necessary to culture the cells in a threedimensional arrangement.(17,18) It is known that a threedimensional arrangement leads to a specific cell shape and
environmental conditions determining gene expression and
behavior of cells.(17) The present work shows that a close
three-dimensional contact of human spherical chondrocytes
enables them to arrange themselves in three-dimensional
cell aggregates, to synthesize cartilage-specific proteins and
matrix components, and to deposit the components in the
intercellular space (Fig. 4). This behavior parallels the natural process of chondrogenesis: aggregation of chondroprogenitor cells followed by the synthesis of a cartilaginous
extracellular matrix.(19) Therefore, it is obvious that the
cellular environment and cell shape of human chondrocytes
in aggregate culture is responsible for this histotypical organization.
A multitude of studies have been done using different
ways to culture chondrocytes in three-dimensional systems.
First of all, various scaffold materials were used to create a
three-dimensional system (e.g., agarose, alginate, collagen,
fibrin glue, polyglycolic acid [PGA], and polylactide acid
[PLA]).(20 26) All these cell-seeded scaffolds have the
growing of cells in a three-dimensional environment accompanied by a regaining or maintenance of cartilage-specific
features in common. However, with respect to clinical application, naturally occurring xenogenous and allogenous
materials (e.g., type I collagen, hyaluronic acid, or fibrin
glue) may be immunogenic and bear safety risks, and their

IN VITRO ENGINEERING OF HUMAN AUTOGENOUS CARTILAGE

1425

FIG. 4. In vitro engineered human cartilage after 3 months cultured in autologous serum. (A)
Live tissue (1.8 1.5 0.5
mm). (B) Safranin O staining:
cells in the core are widely separated by extracellular matrix
mainly consisting of hyalinecartilage specific proteoglycans
(arrow). In the outer regions, the
matrix deposition is reduced (arrowhead). (CE) Immunolocalization of different proteins counterstained with hematoxylin: (C)
collagen type II, (D) S-100 protein, and (E) collagen type I in the
outer region and the inner part of
the aggregate. (F) HE staining of
native cartilage.

application is partially forbidden by the Drug Act. One

attempt to resolve these problems is the use of atelocollagen, where the antigenic determinants on the peptide chain
of type 1 collagen (telopeptide) are removed.(9,11) The phenotype of freshly isolated chondrocytes could be maintained
in the atelocollagen gel, and a cartilage-like tissue developed after implantation in rabbit and in human. However,
L-ascorbic acid is necessary to culture the cell-seeded scaffolds, and patients have to be tested concerning their allergic
reaction to atelocollagen. Furthermore, chondrocytes were
directly seeded into the gel after isolation from the biopsy,
wherefore a rather large cartilage specimen has to be harvested from healthy cartilage tissue from the patient. Other
scaffolds are not biodegradable or are resorbed with a
greater time constant than cartilage regeneration (e.g., hyaluronic acid).(27) During this resorption process, polymer
scaffolds may produce harmful degradation products.(28)
Furthermore, the integration of the cell-seeded scaffold to
the adjacent normal cartilage is sometimes not shown or
often incomplete.(11,14,29)

With the aim to avoid scaffold materials for threedimensional culture, chondrocytes were grown in micromass or high-density systems. However, cartilage-like morphology and reexpression of cartilage-specific proteins
could only be maintained for up to 4 weeks and only in the
presence of transforming growth factor 1 (TGF-1), bone
morphogenetic protein (BMP)-2, or ascorbic acid.(30 36)
Additionally, most of the growth factors are not permitted
for the processing of human cell based drugs. Using our
described autologous spheroid culture system, neither the
addition of scaffolds, growth factors, or cytokines nor physical manipulations were necessary to induce the formation
of stable cell aggregates and the specific chondrogenic
phenotype of cells.
The in vitro generated cartilage-like tissue is characterized by a time-dependent increased expression of collagen
type II, S-100, and cartilage-specific proteoglycans, paralleled by a reduction of the cell-matrix-ratio. This indicates
a progressive phenotypical differentiation of chondrocytes
and a potential for matrix maturation. The extracellular

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ANDERER AND LIBERA

FIG. 5. Chondrocyte aggregates after different time points in

FCS culture. (A) Five-day-old
aggregate still with a hole in the
core. (B) Four-week-old aggregate with only 1-week expression
of collagen type II in the interior.
(C and D) Immunolocalization of
different proteins in 3-month-old
aggregates. (C) S-100 protein
only expressed in the vital rim
and (D) collagen type I expressed
in the vital rim and the merging
zone.

matrix deposition starts in the core of the spheroids

(Fig. 4B), resulting in a gradient of matrix deposition. This
initial pattern of matrix deposition is also observed in studies using chondrocyte-seeded scaffolds, where the gradient
is equalized in long-time culture.(18) Independent of culture
time, the locally different matrix production in spheroids is
paralleled by a heterogenous cell morphology comparable
with native cartilage tissue: round cells in deeper regions
and flattened cells in the outer zone.(37) This stable hyalinelike in vitro tissue morphology indicates optimized tissue
culture conditions.
Keeping in mind that tissue engineering based therapies
necessitate high amounts of cells, but only small biopsy
specimens with a low yield of cells are available, an augmentation of cell number before a tissue engineering process is unavoidable. Additionally, transferring a small
amount of freshly isolated chondrocytes directly into a
three-dimensional system leads to a proliferation stop (see
Results), resulting in an insufficient cell number and density
for tissue regeneration processes.(38) Expanding chondrocytes in monolayer culture results in the loss of their
cartilage-specific phenotype and matrix protein expression,
and a modified cell behavior (e.g., responsiveness to growth
factors).(39 42) Our experiments also show that human
chondrocytes cultured as a monolayer shift their collagen
expression from type II to type I, paralleled by a loss of the
intracellular protein S-100. However, even after seven passages in monolayer culture, chondrocytes restored their
cartilage-specific phenotype after transferring into autologous three-dimensional culture. The reexpression of collagen type II shows that the shift in collagen expression is
only a transient phenotype. Therefore, the monolayer culture seems to be a useful tool for cell expansion.(43)

To increase the size of in vitro cartilage-like tissue, the

initial cell number could be changed or the ability of aggregates to coalesce could be used. From work done in
tumor spheroid cultures, it is known that nutrient diffusion
is not sufficient to maintain viability of core cells in spheroids larger than 800-1000 m in diameter.(44 46) For that
reason, this study focused on aggregates smaller than 800
m in height. When smaller aggregates were fused, the
fusion process was mediated by the flattened surface cells
(Fig. 2A). Morphological changes of chondrocytes accompanying the coalescence of spheroids indicate the potential
of the cells to adapt to changed environmental conditions.
Furthermore, the migration of outer spheroid chondrocytes
on artificial surfaces, as well as on native tissue, showed a
potentially capacity of spheroids to adhere and integrate
with appropriate structures such as cartilage. This integrative property of spheroidal in vitro cartilage fulfills one of
the major challenges confronting in vitro engineered tissues in regenerative medicine.
Standard cell culture procedures use FCS as medium
supplement because of the limited availability of human
autologous serum. After replacing autologous serum by
FCS in the spheroid culture system, aggregates failed to
produce stabile compact spheroids with the chondrocytespecific phenotype. Neither long-time stability of aggregates, viability of core chondrocytes, nor cartilage-specific
protein expression could be maintained (Figs. 5C and 5D).
It is assumed that the deviating composition and/or amount
of serum components(47) (e.g., in regard to growth factors,
hormones, or further xenogenous proteins) are responsible
for the altered aggregation, differentiation, and finally the
death of human chondrocytes (Fig. 5).

IN VITRO ENGINEERING OF HUMAN AUTOGENOUS CARTILAGE

FIG. 6. Three-week-old chondrocyte aggregates after different time

points in co-culture with human condyle from an osteoarthritic patient
(HE staining). (A) After 45 minutes, multiple focal adhesion points
were formed, and surface cells of the aggregate have already changed
their morphology from flattened to spherical cells (arrows). (B) After
18 days, the aggregates became flattened; new matrix was synthesized.
This new cell-rich matrix (filled arrowhead) differs from condyle
cartilage because it has lower cell density (open arrowhead). Cells of
the in vitro aggregates integrated well into the in vivo condyle cartilage
(arrows).

Further standard supplements in cell culture systems are

antibiotics to suppress bacterial contamination. The addition
of antibiotics to autologous/allogenous culture medium allowed cells in spheroids to survive, but a higher cell/matrix
ratio was displayed compared with those cultures maintained in the absence of antibiotics. This inhibited differentiation of chondrocytes may be caused by the inhibition of
protein synthesis by streptomycin or an effect of penicillin
on extracellular matrix deposition.(48) Taking together, these
results show that in vitro three-dimensional explant or tissue
engineering studies using human cells or tissues may be
optimized by the presence of donor-specific serum and the
absence of antibiotics. However, the availability of donorspecific serum is limited. Our results also showed that the
supplementation of cell culture medium with pooled human
serum is a suitable alternative for in vitro tissue engineering
studies.
Prior uses of the three-dimensional spheroid culture system are well established in tumor biology, where cells are
cultured as multicellular tumor spheroids (MTS). MTS were
developed to maintain tumor cell physiology in vitro that is

1427

altered in monolayer culture.(49,50) Important differences

between MTS and nontumorigenic spheroids are the proliferation and invasion into neighboring tissue. Tumor cells in
the rim of the MTS continue to divide, resulting in a
continuous growth of spheroids. In contrast, proliferation of
human chondrocytes is inhibited when cultured as spheroids. In contrast to MTS cells, cells from chondrocyte
spheroids do not invade adjacent tissue.(51) Results show
that chondrocytes from spheroids seem to have the capacity
to migrate on the surface of tissue explants and recover
osteoarthritic fissures.
This aggregate culture system is a very effective method
to generate in vitro cartilage-like tissue without using any
scaffold, growth factors, or further additives. Using the
aggregate culture technique supplemented only with autologous serum, chondrocytes formed a hyaline-like threedimensional cell-matrix arrangement. The in vitro engineered tissues are characterized by a long-time stability and
show the capacity for integration with native tissue. Reaching a size of approximately 1 mm, the in vitro tissues are
suitable for clinical use, pharmaceutical test systems, and
scientific studies.
At this stage of our investigations, underlying mechanisms of morphology and physiology of cells and matrix
maturation in spheroids are not known. Further studies
should clarify if the different environmental conditions of
surface/core cells and a gradient of nutrients, oxygen, and
metabolics within the spheroids are responsible for this
cartilage engineering process.

ACKNOWLEDGMENTS
We are very grateful to Dr. Tim Ganey (Medical Center,
Atlanta, GA, USA) for discussion and helpful comments
and to Prof. A. Herrmann (Humboldt University of Berlin,
Institute of Biology/Biophysics, Berlin, Germany) for critical discussion and referring the manuscript.

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Address reprint requests to:

Dr. Jeanette Libera
co.don AG
Molecular Medicine, Biotechnology,
and Tissue Engineering
Warthestrasse 21
D-14513 Teltow, Germany
Received in original form August 31, 2001; in revised form March
11, 2002; accepted April 3, 2002.